Chapter 5 Genetic Analysis of Apomixis - cimmyt

36 GaOO F. er...media. There may also be a statisticalcorrelation of optimum refractive index toDNA content per chromosome, with largegenomes tending to require a higher no'For example, Zephyranthes can be clearedin methyl salicylate (no = 1.537), butNothoscordum (which has biggerchromosomes) requires a higher no' asprovided by 2:1 benzyl benzoate: dibutylphthalate (no = 1.542). Possibly thiscorrelates more directly to G-C content,pitch of the double helix, or degree ofcytosine-methylation in heterochromatin,than to total DNA content; the smallergenomes simply carry lessheterochromatin.Table 3.1 presents refractive indices ofvarious published and candidate clearingmedia as measured with an Abberefractometer at ca. 20°e. Most ovuleclearingstudies have used one of twoclasses of media: modified lactophenol(Herr's 4-1/2 and BB 4-1/2, Herr 1971 and1974) and aromatic esters (Crane 1978;Young et al. 1979; Crane and Carman 1987).The latter have been combined successfullywith staining in hemalum (Stelly et aI.1984) or azure dyes (G. L. Hodnett,personal comm.) for brightfieldobservation. The Herr media do not fullydehydrate the ovule because of the 15%water in commercial lactic acid, andorganelles and cell walls therein seem toclear optimally around no 1.51, whereasthe optimum in aromatic esters is aroundno 1.53 or 1.54. A third class, nearlysaturated sugar solutions, has been usedto document callose deposition (Peel et al.1997), with lesser success in displayingnuclear and cellular locations. A fourthclass, salts in glycerol or sugar solutions,remains to be tested, but appropriaterefractive indices have been obtained forhigh-quality clearing (Table 3.1).Both of Herr's 4-1/2 media contain chloralhydrate, a federally controlled substance thatsometimes presents legal problems for institutionsthat possess it. They also contain eugenol, whichturns yellow upon exposure to light, and phenol,which likewise discolors and is a chemical contacthazard. On the other hand, excellent photographshave been obtained with Herr's media inappropriate species, and their refractive indexprobably can be adjusted upward withoutTable 3.1 Refractive index (n D) of (ommon andpotential dearing mediaSubstance20% sucrose 1.36340% sucrose 1.39960% sucrose 1.44170% sucrose (soturated) 1.465saturoted sucrose in 50% glycerol 1.46880% fructose (saturated) 1.48380% fructose in 10M Kacetate, 10 mM KOH 1.49480% fructose in saturated (ca. 8.6 M) KI, 10 mM KOH 1.51795% glycerol saturated with KI, 1mM KOH 1.506saturated CaCl 2in H0 1.4692saturated CaCI 2in glycerol (glassy solid at room temperature) 1.534saturated ZnCI 2in H021.550saturated ZnCI 2in 80% (v:v) glycerol 1.5321:1:4 (v:v:w) water, glycerol, ZnCl 21.518FeCI 36H 20 liquefied with a trace of glycerol 1.56050% polyvinylpyrrolidone (MW 10000) 1.42975% polyvinylpyrrolidone (MW 10000) 1.487saturated naphthalene in glycerol 1.472Herr's 41/2 medium 1.503Herr's BB 41/2 medium 1.510methyl salicylaJe 1.537dibutyl phthalate (DBP) 1.492benzyl benzoate (BB) 1.569BB:DBP mixes, v:v:50:50 1.53055:45 1.53360:40 1.53765:35 1.54170:30 1.54575:25 1.54980:20 1.552saturated KI + 2.5 mg/mllris base in 50% DMSO 1.475saturated KI + 2.5 mg/mllris base in 70% DMSO 1.496saturated KI + 2.5 mg/ml Iris base in 80% DMSO 1.508saturated KI + 2.5 mg/mltris base in 90% DMSO 1.519saturated KI + 2.5 mg/mltris base in DMSO 1.525n D